Methods and devices for reducing the mineral content of vascular calcified lesions

ABSTRACT

Methods and devices are provided for at least reducing the mineral content of a vascular calcified lesion, i.e. a calcified lesion present on the vascular tissue of a host. In the subject methods, the local environment of the lesion is maintained at a subphysiologic pH for a period of time sufficient for the mineral content of the lesion to be reduced, e.g. by flushing the lesion with a fluid capable of locally increasing the proton concentration in the region of the lesion. Also provided are systems and kits for practicing the subject methods. The subject methods and devices find particular use in the treatment of vascular diseases associated with the presence of calcified lesions on vascular tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.09/195,291,now U.S. Pat. No. 6,387,071 filed Nov. 18, 1998; whichapplication is a continuation-in-part of application Ser. No. 09/118,193now U.S. Pat. No. 6,394,096 filed on Jul. 15, 1998; the disclosures ofwhich are herein incorporated by reference.

INTRODUCTION

1. Technical Field

The field of this invention is vascular disease, particularly vasculardiseases characterized by the presence of calcified lesions, e.g.atherosclerosis, and the like.

2. Background of the Invention

The formation of plaques or lesions, (atherosclerotic plaques orlesions) on cardiovascular tissue, such as the inner surface of bloodvessels, aortic valves, etc., is a major component of cardiovasculardisease. Many atherosclerotic plaques and lesions are characterized bythe presence of mineral deposits, i.e. they are calcified. Calcifiedlesion formation on prosthetic devices is also a problem in currentcardiovascular disease treatment protocols. For example, calcificationis an important limitation on the useful life expectancy ofbioprosthetic valves, and accounts for over sixty percent of the cardiacbioprostheses failures.

A variety of different protocols have been developed for treatingcardiovascular diseases associated with the presence of calcifiedlesions. Such treatment methodologies generally involve mechanicalremoval or reduction of the lesion, and include: bypass surgery, balloonangioplasty, mechanical debridement, atherectomy, valve replacement, andthe like. Despite the plethora of different treatment strategies thathave been developed for the treatment of cardiovascular disease, thereare disadvantages associated with each technique, such as tissue damage,invasiveness, etc. For example, restenosis is a common complication thatresults in arteries in which lesions have been mechanically removed.

As such, there is continued interest in the development of new treatmentprotocols for the removal of vascular calcified lesions from vasculartissue. Of particular interest would be the development of a treatmentprotocol that is minimally invasive and/or results in minimal tissuedamage.

Relevant Literature

U.S. Patents of interest include: U.S. Pat. Nos. 4,445,892; 4,573,966;4,610,662; 4,636,195; 4,655,746; 4,824,436; 4,911,163; 4,976,733;5,059,178; 5,090,960; 5,167,628; 5,195,955; 5,222,941; 5,380,284;5,443,446; and 5,462,529.

SUMMARY OF THE INVENTION

Methods for at least reducing the mineral content of a calcified lesionon vascular tissue are provided. In the subject methods, the localenvironment of the target lesion is maintained at a subphysiologic pHfor a period of time sufficient for the desired amount ofdemineralization to occur, e.g. by flushing the lesion with a fluidcapable of locally increasing the proton concentration in the region ofthe calcified lesion. As a result, the mineral content of the calcifiedlesion is reduced. Also provided are kits and systems for practicing thesubject methods. The subject invention finds use in a variety ofdifferent applications, including the treatment of vascular diseasesassociated with the presence of calcified lesions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a cutaway view of a vessel being treated according toone embodiment of the subject invention.

FIG. 2 provides a cutaway view of a vessel being treated according to asecond embodiment of the subject invention.

FIG. 3 provides a cutaway view of a vessel being treated according to athird embodiment of the subject invention.

FIG. 4 provides a cutaway view of a vessel being treated according to afourth embodiment of the subject invention.

FIG. 5 provides a cutaway view of a vessel being treated according to afifth embodiment of the subject invention, in which the target lesiontotally occludes the host vessel.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods are provided for at least reducing the mineral content of acalcified lesion on vascular structure, e.g. vascular tissue, vascularprosthetic implant, etc. In the subject methods, the local environmentof the calcified lesion is maintained at a subphysiological pH for asufficient period of time for the desired amount of demineralization tooccur, e.g. by flushing the lesion with a fluid capable of locallyincreasing the proton concentration in the region of the lesion. Thesubject methods find use in the treatment of vascular diseasescharacterized by the presence of calcified vascular structure calcifiedlesions. Also provided are kits and systems for use in performing thesubject methods. In further describing the subject invention, thesubject method is discussed first, both in general terms and in terms ofspecific representative applications. This discussion is then followedby a description of systems and kits for use in practicing the subjectmethods.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

It must be noted that as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise. Unless defined otherwiseall technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs.

Methods

The invention provides a method for at least reducing the mineralcontent of a vascular calcified lesion by contacting the lesion with afluid capable of locally increasing the proton concentration in theregion of the lesion. As used herein, the term “vascular” is usedbroadly to refer to the circulatory system of an organism. As such, theterm “vascular” refers to arteries and veins, as well as specializedorgans that are closely associated with the circulatory system, such asthe heart. The term “cardiovascular” refers to that portion of thevascular system that is closely associated with the heart. Thus, targetlesions of the subject methods are vascular calcified lesions, includingcardiovascular calcified lesions.

A lesion is considered to be a vascular calcified lesion if it ispresent on a vascular structure. Vascular structures include vasculartissues as well as vascular implants positioned within the vascularsystem. Vascular tissue refers to any tissue that is present in thecirculatory system of the host, as described above, and as such includesnot only vessel tissue, such as arterial and venous tissue, but alsocardiac or heart tissue, including valves and other cardiovascularfeatures or specialized tissue structures. Vascular implants includeprosthetics that have been introduced into the vascular system,including bioprosthetics, etc, such as allogeneic and xenogeneicimplants, e.g. heart valves, synthetic implants, vascular replacementsor grafts, e.g. saphenous vein grafts, artificial hearts, leftventricular assist devices, electrodes, and the like. Thus, vascularstructures include both naturally occurring vascular tissue and implantsof exogenous origin that have been introduced into the circulatorysystem.

The vascular structure on which the target calcified lesion is presentis a structure found on the blood side of the circulatory system, bywhich is meant that the structure is found on the side of thecirculatory system adjacent to blood flow and which comes into contactwith blood, and not on the outside of the circulatory system, i.e. thatportion of the circulatory system that does not contact blood. As such,the lesion may be present on: (a) the inner wall or intima of a bloodvessel; (b) a valve present in a blood vessel; (c) a heart valve; (d) animplant present in an artery or vein; etc.

Calcified Target Lesion

The calcified target lesion may be a substantially pure mineral depositor coating over the surface of a region of vascular tissue, such as acoating or layer on at least a portion of valve tissue and the like, ormay be a more complex formation that includes both mineral and othercomponents, including organic matter, e.g. lipids, proteins, and thelike.

The mineral component making up the calcified lesion is generally madeup of one or more calcium phosphates, where the calcium phosphates aregenerally apatitic. The term “apatite” as used herein refers to a groupof phosphate minerals that includes ten mineral species and has thegeneral formula X₅(YO₄)₃Z, where X is usually Ca²⁺ or Pb³⁺, Y is P⁵⁺ orAs⁵⁺, and Z is F⁻, Cl⁻, or OH⁻. The term calcium apatite refers to agroup of phosphate minerals where X is Ca²⁺. The mineral component ofthe calcified lesion typically includes one or more of hydroxyapatite,carbonated hydroxyapatite (dahllite) and calcium deficienthydroxyapatite.

In addition to the mineral component, the lesion that is the target ofthe subject methods may also comprise one or more additional components,where such components include: lipids; lipoproteins; proteins; includingfibrinogen, collagen, elastin and the like; proteoglycans, such aschondroitin sulfate, heparin sulfate, dermatans, etc.; and cells,including smooth muscle cells, epithelial cells, macrophages andlymphocytes. As such, calcified lesions that are targets of the subjectmethods include: type IV, type V and type VI lesions, as defined inStary et al., Arterioscler Thromb Vasc Biol. (1995)15:1512-1531.

In arterial lesions that are targets of the subject methods, the mineralcomponent of the calcified lesion generally makes up from about 10 to100, usually from about 10 to 90 and more usually from about 10 to 85dry weight % of the lesion. The size of the lesion that is the target ofthe subject methods varies depending on whether it is a lesion found inarteries, in the aorta or on a valve, e.g. a heart valve. As such, thesize of the lesion may vary substantially, but will typically cover anarea, e.g. surface of arterial intima, of at least about 1 mm², usuallyat least about 4 mm² and more usually at least about 10 mm², where thearea covered by the lesion may be as large as 40 mm² or larger, but willusually not exceed about 20 mm², and more usually will not exceed about15 mm².

Maintaining the Local Environment of the Lesion at a Subphysiologic pH

As summarized above, the mineral content of vascular calcified targetlesions (as described above) is reduced according to the subjectinvention by maintaining the local environment of the lesion at asubphysiological pH for a sufficient period of time for the desiredamount of demineralization to occur. By local environment of the lesionis meant the immediate vicinity of the lesion, such as the area definedby a set distance from any surface point (i.e. point not adjacent orjuxtaposed to the vesicular tissue, e.g. intima, with which the lesionis associated) on the lesion, typically extending at least 1 mm²,usually at least 2 mm² beyond the area covered by the lesion, and inmany embodiments substantially further beyond the area covered by thelesion. For example, where the target lesion covers a 4 mm² surface ofarterial intima, the local environment will extend to cover an area of 6mm². In three-dimensional terms, where a lesion occupies a volume of 8mm³, the volume of the local environment will be at least 9 mm³ and willoften be larger. In many embodiments, the local environment may extendbeyond this limited area. For example, the local environment may be amechanically isolated section of a vessel or valve in which the lesionsare present, where the volume of such an isolated section may range fromabout 4 to 4000 mm³, usually from about 40 to 2000 mm³ and more usuallyfrom about 100 to 1000 mm³. Furthermore, the local environment may be anisolated limb or portion thereof. In yet other embodiments, the localenvironment may be a given length of a blood vessel, e.g. an artery,that has been cannulated on either side of the lesion (e.g. in thoseembodiments where the target lesion is a diffuse lesion that extends fora given length of the blood vessel). In certain embodiments, the volumeof the local environment of the lesion ranges from about 1 to 100,usually from about 5 to 50 and more usually from about 10 to 20 foldgreater than the volume of the lesion, where the local environmentvolume includes the volume of the lesion. In other embodiments, thelocal environment includes a defined area adjacent to only one side ofthe target lesion, e.g. where the target lesion is a substantiallycomplete vascular occlusion. In such embodiments, the local environmentwill not necessarily be larger that the total volume of the targetlesion, but will instead merely include the region of the vessel volumeadjacent to one surface of the vascular occlusion. Importantly, however,the local region does not include the entire vascular system. As such,the local environment of lesion is less than 90%, usually less than 80%and more usually less than 50% of the entire volume (e.g. the volume ofcirculating blood) of the vascular system of the host or subject beingtreated. In many embodiments, the local environment is less than 5% andtypically between about 1 to 2% of the entire volume of the vascularsystem of the host.

Preferably, the local environment of the lesion is at leastsubstantially bloodless, by which is meant that the local environmentcontains substantially no blood components, particularly red bloodcells, white blood cells, platelets, serum proteins, e.g. albumin, andthe like. By substantially bloodless is meant that the local environmentincludes less than 75%, usually less than 50% and more usually less than25% of the blood components originally present in the local environment(where percentage is based on dry weight), where the number oforiginally present blood components in the local environment ispreferably less than 20%, more preferably less than 15% and mostpreferably less than 10%. The local environment is renderedsubstantially bloodless using any convenient methodology, whererepresentative methodologies are provided infra.

As mentioned above, the pH in the local environment is maintained at asubphysiological level for a sufficient period of time for the desiredamount of demineralization of the target lesion to occur. Typically, thepH is maintained at a value that does not exceed about 5 and usuallydoes not exceed about 4, and more usually does not exceed about 3. Inmany embodiments, the pH of the dissolution solution ranges from between0 and 1. Within the above range, the pH may be constant or variable overthe course of the demineralization procedure, i.e. over the period oftime during which the pH of the local environment is maintained at asubphysiological value.

The time period during which the local pH is maintained at asubphysiological level in the local region of the lesion is sufficientfor the desired amount of demineralization to occur. As such, the pH ofthe local environment is maintained at a subphysiological value for aperiod of time ranging from about 5 to 200 minutes, usually from about10 to 100 minutes and more usually from about 10 to 30 minutes.

The pH of the local environment in the region of the lesion may bemaintained at the requisite subphysiological level using any convenientprotocol. Where a substantially constant subphysiological level isdesired, a dynamic introduction of the fluid into the local environmentis employed. Alternatively, where some variability in the pH of thelocal environment is acceptable, a static introduction of the fluid intothe local environment may be employed. Dynamic and static introductionmethods are described in greater detail infra. Of particular interest inmany embodiments is the use of a dissolution solution that is introducedinto the local environment of the lesion and is capable of locallyincreasing the proton concentration in the local environment of thelesion. By capable of locally increasing the proton concentration ismeant that the dissolution solution, upon introduction into the localenvironment of the lesion, as described in greater detail below, iscapable of increasing the hydrogen ion concentration or [H⁺] in theregion of the lesion. In other words, the solution is capable ofreducing the pH in the region of the lesion to the requisitesubphysiologic level for the required demineralization to occur.

As mentioned above, in preferred embodiments, the local environment ofthe lesion is substantially, if not completely, bloodless. As such, themethod of the subject invention typically includes a step of renderingthe local environment of the lesion at least substantially bloodless.Any means of rendering the local environment bloodless may be employed,such as the use of devices with balloons, cannulation devices, and thelike, where representative methods of rendering the local environment ofthe target lesion substantially bloodless are described in furtherdetail infra.

Dissolution Solutions

A variety of different types of dissolution solutions may be employed inthe subject methods, as long as the solutions are capable of increasingthe proton concentration locally in the region of the target lesion tothe desired subphysiologic level. In other words, any solution that iscapable of providing the requisite subphysiologic pH in the localenvironment of the lesion is suitable for use in the subject methods.Instead of using a single dissolution solution, a plurality of differentdissolution solutions which vary by one or more parameters (e.g. type,pH, concentration etc.) may be sequentially introduced into the regionof the lesion. In such embodiments, the number of different dissolutionsolutions employed is at least 2, but generally does not exceed about 4and usually does not exceed about 3.

One type of solution that finds use is an acidic dissolution ortreatment solution. The acidic treatment solution will generally have apH of less than about 6.5, where the pH is usually less than about 4.0and more usually less than about 3.0. In many preferred embodiments, thepH ranges from 0 to 2, and usually 0 to 1. The acidic treatment solutioncan include a number of different types of acids, where the acids may ormay not include a hydrocarbon moiety, i.e. a hydrogen bonded directionto a carbon atom. Suitable acids that lack a hydrocarbon moiety includehalogen acids, oxy acids and mixtures thereof, where specific acids ofinterest of this type include, but are not limited to, hydrochloric,nitric, sulfuric, phosphoric, hydroboric, hydrobromic, carbonic andhydroiotic acids. For such acids, the acid can be a concentrated acid,or can be diluted. Upon dilution, the concentration of an inorganic acidwill generally be from about 10 N to about 0.01 N, preferably between 5N to 0.1 N. Also of interest are acids that include a hydrocarbonmoiety, where such acids include, but are not limited to, any organicacid of one to six (C₁ to C₆) carbons in length. Organic acids of thistype include, but are not limited to, formic, acetic, propionic, maleic,butanoic, valeric, hexanoic, phenolic, cyclopentanecarboxylic, benzoic,and the like. For an organic acid, the acid can be in concentrated form,or can be diluted. The acidic treatment solution can be composed ofeither a monobasic or a polybasic acid. Acids are “monobasic” when theyhave only one replaceable hydrogen atom and yield only one series ofsalts (e.g., HCl). Acids are “polybasic” when they contain two or morehydrogen atoms which may be neutralized by alkalies and replaced byorganic radicals.

In many embodiments of the subject invention, the acid solution ishypertonic, by which is meant that the osmolarity of the solution isgreater than that of a red blood cell, i.e. the osomolarity is greaterthan 300 mosmol. The solution may be rendered hypertonic by includingany convenient component or components in the solution which provide forthe desired elevated osmolarity.

Any convenient agent that is capable of increasing the osmolarity of thesolution may be employed, where suitable agents include salts, sugars,and the like. In many embodiments, the agent that is employed to renderthe solution hypertonic is one or more, usually no more than three, andmore usually no more than two, different salts. Generally, the saltconcentration in these embodiments of the solution is at least about 100mosmol, usually at least about 200 mosmol and more usually at leastabout 300 mosmol, where the concentration may be as high as 3000 mosmolor higher, depending on the particular salt being employed to render thesolution hypertonic, where the solution may be saturated with respect tothe salt in certain embodiments. Salts that may be present in thesubject solutions include: NaCl, MgCl₂, Ringers, etc. where NaCl ispreferred in many embodiments.

Two acid solutions of particular interest are hydrogen chloridesolutions and carbonic acid solutions. Each of these is discussed ingreater detail below.

Hydrogen Chloride Solutions

Hydrogen chloride solutions finding use in the subject methods have anHCl concentration that is sufficient to provide for the requisite pH inthe local environment of the target lesion. Generally, the concentrationof HCl in the solution ranges from about 0.001 to 1.0 N, usually fromabout 0.01 to 1.0 N and more usually from about 0.1 to 1.0 N. In manyembodiments, the hydrogen chloride solution will further include one ormore salts which make the solution hypertonic, as described above. Incertain preferred embodiments, the salt is NaCl, where the concentrationof NaCl in the solution is at least 0.05 M, usually at least 0.10 M, andmore usually at least 0.15 M, where the concentration may be as high as0.25 M or higher. In certain embodiments, the solution will be saturatedwith NaCl.

Carbonic Acid Solutions

In another preferred embodiment of the subject invention, the solutionthat is employed is a carbonic acid solution. Carbonic acid solutionsthat find use are aqueous solutions that have a pH that is sufficientlylow to achieve the desired subphysiological pH in the local region ofthe lesion during treatment. As such, the pH of the carbonic acidsolution is typically less than about 6, usually less than about 5 andmore usually less than about 4, where the pH may be as low as 2 orlower, but will generally not be below about 1. The carbonic acidconcentration of the solution may vary, but will generally range fromabout 0.1 to 4.0 M and usually from about 0.1 to 1.0 M. The carbonicacid solution should be bubble free, i.e. CO₂ bubble free, during use.As such, the pressure and/or temperature of the carbonic acid solutionmay be modulated to provide the requisite bubble free properties. Thecarbonic acid solution may be at ambient or elevated pressure, i.e.pressurized. Where the carbonic acid solution is pressurized, it will bepressurized to at least about 10 bar (10 atm), usually at least about 50bar and more usually to at least about 100 bar, where it may bepressurized to a pressure of 1000 bar or greater. The temperature of thecarbonic acid solution may vary from about 0 to 37° C., usually fromabout 10 to 37° C. and more usually from about 20 to 37° C.

The carbonic acid solution may be produced in a number of differentways. For example, the carbonic acid solution may be prepared bycombining sodium bicarbonate and hydrogen chloride solutions in a mannersufficient to produce a carbonic acid solution. The sodium bicarbonatesolution that is employed will generally have a sodium bicarbonateconcentration ranging from about 0.01 to 1.0 M, and usually from about0.02 to 0.1 M. The hydrogen chloride solution that is employed will havea concentration ranging from about 0.01 to 1.0 M and usually from about0.01 to 0.5 M. Upon combination of the sodium bicarbonate solution andhydrogen chloride solution, carbonic acid is produced in accordance withthe following equilibrium equation:

NaHCO₃+HCl═H₂CO₃+Na⁺+Cl⁻═H⁺+HCO₃ ⁻

The equilibrium of the above reaction is maintained in favor ofproduction of the proton by maintaining the pressure and temperature ofthe solution at appropriate values. For a solution prepared in thismanner, the pressure of the solution is maintained in a range of fromabout 10 to 200 bar and usually from about 50 to 150 bar while thetemperature is maintained at a value ranging from about 0 to 37° C. andusually from about 20 to 37° C.

The carbonic acid solution that finds use in the subject invention canalso be produced by making an aqueous solution that is saturated withrespect to CO₂. In this embodiment, the solution is maintained as bubblefree, by which is meant that CO₂ gas is prevented from coming out ofsolution such that the carbonic acid equilibrium reaction:

CO₂+H₂O═H₂CO₃+Na⁺+Cl⁻═H⁺+HCO₃ ⁻

is driven in the direction of carbonic acid, i.e. H₂CO₃, andconsequently proton and bicarbonate ion production. Generally, the pCO₂in this carbonic acid solution is at least about 100, usually at leastabout 500 and more usually at least about 1000 mmHg, where the pCO₂ ofthe solution may be as high as 5000 mmHg or higher, but will generallynot exceed about 10,000 mmHg. The solution prior to delivery willtypically be pressurized to some pressure above atmospheric pressuresuch that it remains bubble free and yet saturated, even supersaturated,with respect to the CO₂. As such, the pressure of the solution isgenerally at least about 10 bar, usually at least about 50 bar and moreusually at least about 100 bar, where the pressure may be as high as 200bar or higher, but will generally not exceed about 1000 bar. Thetemperature of the solution may also be modulated to obtain the desireddissolved CO₂ in the solution. As such, the temperature may range fromabout 0 to 37° C., usually from about 10 to 37° C. and more usually fromabout 20 to 37° C. A variety of technologies are known to those of skillin the art for producing aqueous solutions that are saturated withrespect to CO₂, any of which may be employed to produce the carbonicacid solution finding use in the subject methods. Of particular interestare the techniques disclosed in U.S. Pat. Nos. 5,086,620; 5,261,875;5,407,426; 5,599,296; 5,569,180; 5,693,017; 5,730;935; 5,735,934; and5,797,874; the disclosures of which applications are herein incorporatedby reference. Briefly, a stable, bubble-free saturated CO₂ aqueoussolution is produced by contacting gaseous CO₂ with an aqueous carriermedium, e.g. pure water, under elevated pressure conditions such thatthe gaseous CO₂ goes into, and is maintained in, solution.

Additional Components

The dissolution solutions employed in the subject invention may alsocomprise one or more additional components that serve a variety ofpurposes. Components that may be included are ions which serve to: (a)prevent apatite formation, (b) prevent apatite reformation, (c) modifyapatite solubility, etc., where such ions include Mg²⁺, and the like.When present, the concentration of the magnesium ion in the solutionwill generally range from about 0.01 to 0.20 M, usually from about 0.05to 0.1 M.

The solution may further include an oxygenating medium for delivery ofoxygen to the local environment of the lesion during treatment, i.e. thesolution may further comprise oxygen—the solution may be supersaturatedwith respect to O₂. When present, the solution will comprise 1 to 4,usually 1 to 3 ml O₂/g fluid. Any convenient oxygenating medium may beemployed, including the hyperbaric oxygen mediums disclosed in U.S. Pat.Nos. 5,086,620; 5,261,875; 5,407,426; 5,599,296; 5,569,180; 5,693,017;5,730;935; 5,735,934; and 5,797,874, the disclosures of which are hereinincorporated by reference. An example of situations where oxygenatingmediums find use in the dissolution solution include the treatment ofdiffuse arterial lesions by the subject methods, e.g. diffuse arteriallesions found in the limbic extremities. For example, to treat a lowerlimbic extremity diffuse arterial lesion, e.g. an arterial lesionpresent below the knee, one can produce an isolated local environment byblocking the appropriate artery (e.g. posterior tibial artery, anteriortibial artery) and vein (e.g. great saphenous vein, small saphenousvein) on either side of the diffuse lesion. The lesion can then becontacted, e.g. flushed, with the dissolution solution comprising theoxygenating medium by introducing the solution into the artery andremoving it from the vein, as described in greater detail below. In thisembodiment, the entire circulatory system below the substantiallyblocked portions of the artery and vein is transformed into the localenvironment of the lesion in which a subphysiologic pH is maintained.The oxygenating medium serves to maintain the requisite oxygen levels inthe tissue of the local environment of the lesion.

The dissolution treatment solution can further include calcium-chelatingagents, for example, EDTA, crown ethers, and the like. The concentrationof these agents will vary, but will generally not exceed about 4.0 M andusually will not exceed about 1.0 M.

The dissolution solution may also include an enzymatic component thatserves to promote the formation of protons in the solution and localenvironment of the lesion in order to provide for the subphysiologic pH.A variety of enzymes or activities may be employed, depending on thespecific nature of the dissolution solution. For example, in thoseembodiments in which the dissolution solution is saturated with CO² gas,the solution can further include carbonic anhydrase. The enzyme may be anaturally occurring enzyme or synthetic homologue thereof, where theenzyme may be produced via purification from naturally occurring sourcesor through recombinant technology.

In addition, the dissolution solution may further include one or morecomponents which act on the non-mineral phase of the target lesion inorder to disrupt the lesion and promote its disruption and/ordissolution. Such, organic disruption/dissolution agents that may bepresent in the dissolution solution include: thrombolytic agents, e.g.urokinase, tPA, etc.; enzymes, e.g. proteases, collegenases; heparin;surfactants; detergents; etc.

Contacting the Calcified Target Lesion with the Dissolution Solution

As mentioned above, in the subject methods the dissolution solution isintroduced into the local environment of the lesion in a mannersufficient to maintain the pH of the local environment of the lesion atthe requisite subphysiological level for a sufficient period of time forthe desired amount demineralization to occur. As such, the subjectmethods generally involve contacting the lesion with the dissolutionsolution. The manner in which contact is achieved-may be static ordynamic. By static is meant that a predetermined amount of dissolutionsolution is introduced into the local environment of the lesion andmaintained in the local environment of the lesion for the entiretreatment period, without the addition of further quantities ofdissolution solution. By dynamic is meant that the dissolution solutionis introduced into the local environment of the lesion one or moretimes, including continuously, during the treatment period. As mentionedabove, the local environment of the lesion has preferably been renderedbloodless prior to introduction of the dissolution fluid.

During the dissolution procedure, protons from the local environment areremoved as a result of the demineralization process. As such, it isoften desirable to introduce the dissolution solution into the localenvironment of the lesion in a dynamic manner. Dynamic introduction ofthe dissolution solution typically involves flushing the lesion with thedissolution solution, where flushing involves a continuous flow of thedissolution solution across at least a surface of the lesion, where theflow may be under pressure (e.g. where the fluid is emitted from thedelivery device under enhanced pressure, as described in greater detailinfra). In other words, the dissolution fluid is continuously flowedthrough the local environment of the lesion for the period of timerequired for the desired amount of demineralization to occur.Simultaneously, fluid is removed from the local environment of thelesion such that the overall volume of fluid in the local environment ofthe lesion remains substantially constant, where any difference involume at any two given times during the treatment period does notexceed about 50%, and usually does not exceed about 10%. In this manner,the pressure of the localized environment of the lesion is maintained ata substantially constant value, thereby minimizing traumatic impact onthe vessel walls in the region of the lesion.

Where the lesion is flushed with the dissolution solution, the flow rateof the dissolution solution through the local environment of the lesionis generally at least about 1 volume/minute, usually at least about 2volumes/minute and more usually at least about 10 volumes/minute, wherethe flow rate may be as great as 100 volumes/minute or greater, butusually does not exceed about 1000 volumes/minute and more usually doesnot exceed about 500 volumes/minute, where by “volume” is meant thevolume of the local environment of the lesion.

When treatment involves dynamic flushing of the local environment of thelesion, the total amount of dissolution fluid that is passed through thelocal environment of the lesion during the treatment period typicallyranges from about 0.5 to 50 liters, usually from about 0.5 to 5.0 litersand more usually from about 0.5 to 2.0 liters. In contrast, where astatic methodology is employed, the total amount of dissolution fluidthat is introduced into the local environment of the lesion ranges fromabout 100 ml to 1 liter, and usually from about 100 to 500 ml.

Devices for Contacting the Target Lesion with the Dissolution Solution

Any convenient means may be employed for introducing the dissolutionsolution into the local environment of the lesion. In general, thedissolution fluid introduction means should at least include a means forintroducing dissolution fluid into the local environment of the lesion.Typically, the means is a conduit, e.g. tube, which has an opening atits distal end (i.e. the end that comes closest to the lesion duringuse) and is in fluid communication at its proximal end with a containerholding the dissolution fluid, where the fluid communicationrelationship can be established through direct contact of the lumen withthe container or through one or more connecting means which establishthe requisite fluid communication.

In many embodiments, e.g. where the lesion is flushed with thedissolution solution, contact also includes removal of solution from thelocal environment of the lesion. Any convenient means may be employedfor removing dissolution solution, as well as particles of lesion anddissolved lesion components, from the local environment of the lesion.The fluid removal means may be incorporated into the fluid introductionmeans summarized above or a separate component from the fluidintroduction means. Thus, fluid removal means may be a conduit or vesselwhich is a component of the fluid introduction means, or may be aconduit or vessel on a separate catheter, cannula etc, which ispositioned “downstream” in the direction of blood flow from the targetlesion and the site of introduction of the dissolution fluid.

In many embodiments, the fluid introduction means is a catheter. In manyembodiments, catheters employed in the subject methods include at leastone fluid introduction means for introducing a dissolution fluid to thelocal environment of the lesion and a fluid removal means for removingfluid from the local environment of the lesion. In many embodiments, thecatheter devices of the subject invention also typically include a meansfor isolating the local environment of the target lesion.

As mentioned above, the dissolution fluid introduction means isgenerally a lumen having a proximal end in fluid communication with thedissolution fluid source, e.g. a dissolution fluid reservoir, and anopen distal end capable of being introduced into the local environmentof the target lesion. By “lumen” is meant an elongated vessel having atubular structure with a proximal and distal end, where thecross-sectional shape along the length of structure is generally (thoughnot necessarily) circular, ovoid or some other curvilinear shape. Thedissolution fluid introduction lumen has sufficient dimensions to allowfor the desired flow rate at the site of the target lesion. The exactdimensions for the fluid introduction lumen will vary depending, atleast in part, on the nature of the dissolution fluid that is to beintroduced in the region of the lesion. For example, with HCl solutions,fluid introduction lumens having inner diameters (ID) ranges from about1 to 5 mm, usually from about 1 to 3 mm and more usually from about 1 to2 mm are typically employed. Alternatively, in those embodiments inwhich a pressurized dissolution fluid is delivered to the localenvironment of the lesion, e.g. where a carbonic acid solution isemployed as the dissolution solution, the dimensions are oftensufficient to reduce bubble formation, e.g. CO₂ bubble formation. Assuch, the dissolution fluid introduction lumen has an inner diameter(ID) that is at least about 50 μm, usually at least about 100 μm andmore usually at least about 200 μm, where the inner diameter willtypically not exceed about 2000 μm and usually will not exceed about1000 μm. Depending on the configuration of the catheter device, theentire cross-sectional area may be available for fluid flow, or aportion of the cross-sectional area may be occupied by one or moreadditional device elements, e.g. a guide wire, one or more additionallumens, and the like, as described in greater detail infra. The fluidintroduction lumen may be fabricated from a wide variety of materials.See the patents listed in the relevant literature section, supra. Inthose embodiments where the dissolution fluid is pressurized, asdescribed above, the lumen is fabricated from materials capable ofpreserving the pressure of the fluid. Such materials are described inU.S. Pat. Nos. 5,599,296; 5,569,180; 5,693,017; 5,730;935; 5,735,934;and 5,797,874; the disclosures of which applications are hereinincorporated by reference. Also of interest are multiple small lumenshaving ID of between about 50 and 80 μm, usually around 75 μm.

In addition to the fluid introduction means, the subject catheterstypically further include a fluid removal means capable of removingfluid from the local region or environment of the lesion. A criticalfeature of the fluid removal means in many embodiments is that it iscapable of removing fluid from the local environment of the lesion atthe same rate as that at which fluid is introduced into the localenvironment of the lesion by the dissolution fluid introduction means.The fluid removal means is typically a lumen having dimensions thatallow for adequate fluid flow from the local environment of the targetlesion. In addition, in certain embodiments the dimensions of the secondlumen are such that they allow passage of the debris from the localenvironment of the lesion through the second lumen. In such embodiments,the fluid removal lumen has an inner diameter that is substantiallylonger than the inner diameter of the fluid introduction lumen, where bysubstantially longer is meant at least about 2 fold longer, usually atleast about 5 fold longer. As such, the fluid removal lumen typicallyhas an inner diameter that is at least about 1 mm, usually at leastabout 2 mm and more usually at least about 3 mm, where the innerdiameter typically does not exceed about 5 mm and usually does notexceed about 4 mm. The fluid removal lumen may be fabricated from anysuitable material, where a variety of suitable materials are known tothe those of skill in the art.

In many embodiments, the subject device further includes a means forsubstantially isolating the local environment of the lesion from theremainder of the host's circulatory system so that the local environmentcan be rendered substantially, if not completely, bloodless. Bysubstantially isolating is meant that fluid communication between thelocal environment of the lesion and the remainder of the host'scirculatory system is essentially removed—i.e. the local environment ofthe lesion is no longer accessible by fluid from the remainder of thehost's circulatory system or vice versa. Any convenient means may beemployed for isolating the local environment of the lesion. Such meansinclude “cup” components that snugly fit over the lesion and therebyisolate it from the remainder of the circulatory system, dual balloonsystems that inflate on either side of the lesion to isolate the localenvironment, etc.

In addition to the above components, the capillary devices of thesubject invention may further include: (a) one or more additionallumens, e.g. for introducing a rinse or wash fluid to the localenvironment of the lesion; a means for allowing blood to flow throughthe isolated local environment, e.g. a pass through lumen; a means forapplying energy to the lesion, e.g. an ultrasonic means; andvisualization or monitoring means; etc.

All of the above components are conveniently present in a catheterdevice capable of accessing the cardiovascular site of interest. Thecatheter device is capable of operatively communicating with othercomponents and devices necessary for operation of the catheter, such asfluid flow means, fluid reservoirs, power means, pressurized gas supplymeans, and the like, as described below, that are part of the overallsystem employed to practice the subject methods.

Representative Devices for Use in Practicing the Subject Methods

Representative embodiments of dissolution fluid introduction (and incertain embodiments removal) means are now described in greater detailin terms of the figures. FIG. 1 provides a representation of a devicefor use in practicing the invention. Artery 12 (shown in cutaway view)has calcified lesion 14 on its inner surface 16. Catheter 11 ispositioned proximal to the target lesion 14. At the distal end ofcatheter 11 is opening 13 which provides for flow of dissolution fluidfrom the catheter into the local environment of the lesion and opening15 which provides for flow of fluid from the local environment of thelesion into the catheter and out of the patient. Catheter 11 alsoincludes balloon element 17 which is inflated to render the localenvironment of the lesion substantially bloodless. During use, fluidinflow and outflow are kept at substantially equal rates so as tomaintain a substantially constant pressure in the region of the targetlesion. In certain embodiments, the catheter is configured such thatdissolution fluid is forced out of port 13 at high pressure (e.g. as ajet). This embodiment finds particular use in the treatment of occlusivelesions, as described in greater detail infra. See FIG. 5.

FIG. 2 provides a representation of another catheter design that can beemployed to practice the subject methods. In FIG. 2, catheter 24 has twoinflatable balloons 21 and 22 connected by a conduit 23 at its distalend. Catheter 24 also has fluid inflow opening 25 and fluid outflowopening 26 for introducing and removing dissolution fluid from the localenvironment of the target lesion 14. During use, the catheter isinserted and the balloons inflated such that the local environment ofthe target lesion becomes substantially sealed from the remainder of thehost's circulatory system. The local environment is then flushed withdissolution fluid using openings 25 and 26.

FIG. 3 provides a representation of yet another catheter device that hasbeen designed for use in connection with the present invention. Thedevice is designed for use in minimally invasive procedures and in anopen surgical field. The catheter is shown in artery 12 having calcifiedtarget lesion 14. Catheter 31 has a flexible cup 32 secured near thedistal end of the catheter (shown in transparent lines). In oneembodiment, the cup can be folded for insertion into the vessel, andthen expanded at the desired location in the vicinity of the mineralizedarea. A defined area or local environment is created by the contact ofthe cup 32 with the vessel wall 16. The catheter is designed to allowinfusion of the local environment with the dissolution solution. Thecatheter is composed of flexible tubing such that it can be situated atany position along a vessel, and should be sufficiently strong so thatit withstands the pressure created from the both the flow of the acidictreatment solution and the suction generated during the removal of theacidic treatment solution. Cup 32 can be held in place by maintainingthe pressure within the local environment sufficiently below bloodpressure, or optionally by a balloon (not shown) or other means. Anultrasound probe (not shown) may be used to generate ultrasonic energy.

In one embodiment, the catheter 31 is a single lumen catheter. The lumenof the catheter communicates with the interior of the flexible cup 32. Adissolution solution can be applied through the catheter to the localenvironment for the desired time period. Following this time period, thecup is removed, and the dissolution solution is allowed to disperse.Alternatively, a device to create suction can be applied to the moreproximal end of the catheter so that the dissolution solution is drawnaway from the defined area via the single lumen. Similarly, followingtreatment with the dissolution solution the rinsing agent can be appliedthrough the single-lumen catheter, if desired.

In another embodiment, the catheter 31 is a double-lumen catheter, bothof which communicate with the interior of the flexible cup 32. One ofthe lumens allows the infusion of either the dissolution solution or arinsing solution. The second lumen removes the dissolution or rinsesolution. Infusion and suction can be alternated, or the two process canbe applied simultaneously to create a flow of solution.

In yet another embodiment, catheter 31 is a triple-lumen catheter, allof which communicate with the interior of flexible cup 32. In thisembodiment, one of the lumens allows the infusion of the dissolutionsolution, one of the lumens allows the infusion of a rinsing solution,and one of the lumens allow for the application of suction for theremoval of solution.

Referring to FIG. 4, a double cup assembly for use with the presentinvention is shown. The assembly is designed for use in minimallyinvasive procedures and in an open surgical field. In this apparatus,catheter 45 includes first and second expandable cups 41 and 42. Thecups can be placed on either side of a calcified target lesion, such asa calcified valve 18 shown in FIG. 4. This first cup 42 is placed inclose proximity to one side of valve 18. One lumen of the catheterpasses through the opening of the valve 18, as is terminates at secondcup 41, which is placed in close proximity to the opposite side of thevalve. Catheter 45 also include fluid introduction 43 and fluidextraction 44 openings for introducing and removing fluid from the localenvironment bounded by the cups 41 and 42.

Additional catheter devices that may be employed to practice the subjectmethods include those described in U.S. Pat. Nos. 4,610,662; 4,573,966;4,636,195; 4,824,436; 5,059,178; 5,090,960; 5,167,628; and 5,222,941;the disclosures of which are herein incorporated by reference.

Additional Method Steps

In a number of embodiments of the subject methods, the above step ofmaintaining the local environment of the lesion at a subphysiological pHfor a sufficient period of time for demineralization of the targetcalcified lesion to occur is used in conjunction with one or moreadditional method steps in order to achieve the overall mineralreduction in the target lesion. Additional methods steps that may bepresent in the overall process include: rendering the region of thetarget lesion bloodless, contacting the target lesion with a solutiondesigned to remove organic components, washing or rinsing the localenvironment of the target lesion, contacting the treated vascular sitewith one or more active agents, and the like.

Where one or more additional distinct solutions, such as primingsolutions, washing solutions, organic phase dissolution solutions andthe like are employed, as described below, such disparate solutions aregenerally introduced sequentially to the site of the target lesion. Forexample, the target lesion may be contacted with the following order ofsolutions: (1) priming solution to render the local environmentsubstantially bloodless; (2) organic phase dissolution solution, e.g.detergent solution such as cholic acid solution, to remove organicphases from the target lesion; (3) acidic dissolution solution todemineralize the target lesion; and (4) washing solution. Othersequences of solution application can also be employed.

Rendering the Region of the Target Lesion Bloodless

In many preferred embodiments, as described above, the local environmentof the lesion is rendered substantially bloodless prior to introductionof the dissolution fluid. In these embodiments, the local environmentmay be rendered substantially bloodless using a variety of differentprotocols. Typically, a priming solution will be employed in this stepof rendering the local environment bloodless. Examples of primingsolutions that may find use in these embodiments include: water forinjection, saline solutions, e.g. Ringer's, or other physiologicallyacceptable solutions. The priming solution includes an anticlottingfactor in many embodiments, where anticlotting factors of interestinclude heparin and the like. The priming solution can also containchelating agents.

Removal of blood from the local environment with the priming solutioncan be accomplished using any convenient protocol. For example, wherecannulation is employed, e.g. to isolate a stretch of a blood vessel orto isolate a limbic extremity, the local environment of the lesion maybe flushed with a washing solution by introducing fluid through theproximal (upstream) cannula and removing blood from the downstream(distal) cannula. Where the device that is employed to introduce thedissolution fluid further includes a means for substantially isolatingthe local environment of the lesion (e.g. a balloon or a cup asdescribed above), the contacting step of the subject methods furthercomprises a step of substantially isolating the local environment of thelesion from the remainder of the subject's circulatory system. Thisisolation step varies depending on the particular nature of the deviceemployed. Thus, in certain embodiments, isolation includes inflatingballoons at either end of the lesion, thereby substantially isolatingthe local environment of the lesion.

Use of Organic Structure Dissolution Solutions

As mentioned above, in addition to the acidic dissolution solution,certain embodiments of the subject invention include a step ofcontacting the target lesion with a dissolution solution which serves toremove at least a portion of the non-mineral, typically organic, phaseof the target lesion. The nature of this “organic phase dissolutionsolution” varies depending on the nature of the target lesion.Representative active agents that may be present in this organic phasedissolution solution include: oxidizing agents; organic solvents; lipiddissolving agents such as surfactants, e.g. TWEEN™, and detergents,where ionic detergents are of particular interest, e.g. cholic acid,glycocholic acid, benzylkonium chloride; enzymes, and the like.

Basic Solutions

In one embodiment, the priming solution is a basic solution. The basicsolution can be composed of any inorganic or organic base. The basicsolution can be a concentrated base, or can be a dilute basic solution.The pH of the basic solution is generally greater than about 9.0. In oneembodiment, the basic solution has a pH between about 10.0 and about12.0. The basic solution can be a solution of an inorganic base. In oneembodiment, the basic solution is a solution of sodium hydroxide (NaOH).In one embodiment, the basic solution is a dilute solution of sodiumhypochlorite.

Washing

In most embodiments, it is desirable to rinse or wash the localenvironment of the lesion following treatment with the dissolutionsolution. The rinsing solution can be any solution sufficient to removeor dilute the acidic treatment solution from the vascular tissue,thereby reducing the acidity in the local environment of the lesion. Inone embodiment, the rinsing solution is a neutral solution. The solutionmay include an anticlotting factor, such as heparin. The neutral rinsingsolution can be a buffered solution of physiological pH. Preferably, theneutral rinsing solution has a pH of about 7.0 to about 8.0. Morepreferably, the neutral rinsing solution has a pH of about 7.4. Onenon-limiting example of a neutral rinsing solution is phosphate bufferedsaline.

Lesion Inhibition Agents

In certain embodiments, it is of interest to further treat the localenvironment of the lesion, i.e. which may or may not comprise any of theoriginally present lesions, depending on the particular methodconducted, with one or more agents that serve to inhibit the formationof the new calcified lesion on the vascular tissue on which the lesionwas present. Inhibition agents that may be employed include:water-soluble phosphate esters (e.g., sodium dodecyl hydrogen phosphate,as described in U.S. Pat. No. 4,402,697, the disclosure of which isherein incorporated by reference); water soluble quaternary ammoniumsalts (e.g., dodecyltrimethyammonium chloride, as described in U.S. Pat.No. 4,405,327, the disclosure of which is herein incorporated byreference); sulfated higher aliphatic alcohols (e.g., sodium dodecylsulfate, as described in U.S. Pat. No. 4,323,358, the disclosure ofwhich is herein incorporated by reference); agents that result in thecovalent coupling of aliphatic carboxylic acids (as described in U.S.Pat. No. 4,976,733, the disclosure of which is herein incorporated byreference); and the like. Other agents of interest that may be employedincluding agents of biological origin, such as growth factor inhibitors,angiogenisis inhibitors and the like.

In certain embodiments, the local environment of the lesion is contactedwith a wound healing or growth promoting solution that provides variousgrowth factors to the local environment of the lesion to promote healingof the site. Growth factors of interest include: platelet derived growthfactor, keratinocyte growth factor, basic fibroblast growth factor,leukocyte derived growth factor-2 (LDGF-2), transforming growth factor,epidermal growth factor (EGF), connective tissue growth factor,fibroblast growth factor 11, vascular IBP-like growth factor, epithelialcells growth factor, fibroblast growth factor 13, insulin-like growthfactor-1, vascular endothelial growth factor (VEG-F), and the like.

Application of External Energy

In certain embodiments, external energy is applied to the target lesionto promote mechanical break-up of the lesion into particles or debristhat can be easily removed from the site of the lesion. Any means ofapplying external energy to the lesion may be employed. As such, jets orother such means on a catheter device which are capable of providingvarying external forces to the lesion sufficient to cause the lesion tobreak up or disrupt may be employed. Of particular interest in manyembodiments is the use of ultrasound. The ultrasound can be appliedduring the entire time of contact of the cardiovascular tissue with theacidic treatment solution, or the ultrasound can be applied for onlypart of the treatment period. In one embodiment, ultrasound is appliedfor several short periods of time while the dissolution treatmentsolution is contacted with the cardiovascular tissue. There are severaldevices for the application of ultrasound to cardiovascular tissue knownto those of skill in the art. For example, U.S. Pat. No. 4,808,153, thedisclosure of which is herein incorporated by reference, describes anultrasound apparatus to be used in an artery without damaging theartery, and U.S. Pat. No. 5,432,663, the disclosure of which is hereinincorporated by reference, describes an apparatus for generatingultrasonic energy useful for removal of intravascular blockages. Theultrasound can be low frequency ultrasound.

In such methods where external energy is applied to the lesion in orderto disrupt or break-up the lesion into particles or debris, theparticles or debris may range in size from about 0.01 to 4.0 mm, usuallyfrom about 0.1 to 2.0 mm and more usually from about 0.5 to 1.0 mm. Insuch instances, the method may further include a step in which theresultant particles are removed from the local environment of thelesion. Particles may be removed from the local environment of thelesion using any convenient means, such as the catheter of the subjectinvention described in greater detail infra.

Another means that may be employed to apply external energy to thelesion during the dissolution process is to use a mechanical means ofapplying external energy. Mechanical means of interest include movingstructures, e.g. rotating wires, which physically contact the targetlesion and thereby apply physical external energy to the target lesion.

Imaging

In addition, it may be convenient to monitor or visualize the lesionprior to or during treatment. A variety of suitable monitoring means areknown to those of skill in the art. Any convenient means of invasive ornoninvasive detection and/or quantification may be employed. Such meansinclude plain film roentgenography, coronary arteriography, fluoroscopy,including digital subtraction fluoroscopy, cinefluorography,conventional, helical and electron beam computed tomography,intravascular ultrasound (IVUS), magnetic resonance imaging,transthoracic and transesophageal echocardiography, rapid CT scanning,antioscopy and the like. Any of these means can be used to monitor thereduction in mineralization by the method of the invention.

Demineralization of Calcified Lesions

Maintenance of the local environment of the calcified lesion at asubphysiologic pH, as described above, results in at least partialdemineralization of the lesion, i.e. at least a reduction of the calciumphosphate content of the lesion. By reduction is meant that the totaloverall dry weight of calcium phosphate mineral is reduced or decreased,generally by at least about 50%, usually by at least about 75% and moreusually by at least about 90%. In certain embodiments, substantially allof the calcium phosphate content of the lesion may be removed, where bysubstantially all is meant at least about 90%, usually at least about95% and preferably at least about 99% dry weight of the original calciumphosphate present in the lesion is removed.

Utility

The subject methods find use in a variety of different applications inwhich it is desired to at least reduce, if not substantially remove, atleast the mineral component of a calcified lesion. One application inwhich the subject methods find use is in the treatment of a hostsuffering from a vascular disease associated with the presence ofvascular calcified lesions. Such vascular diseases include diseases inwhich one or more different calcified lesions are present on one or morelocations of the vascular tissue of the host, where the lesion(s) may bepresent on a vessel wall, on a valve, etc.

By treatment is meant at least a reduction in a parameter of thedisease, where parameter may include typical symptoms indicative ofoccluded vessels or malfunctioning valves, e.g. chest pains, angina,limb ischemia, etc., or risk factors associated with the disease orcondition, e.g. narrowing of arteries, and the like. Treatment alsoincludes situations where the host is cured of the vascular disease,i.e. where the lesion is completely removed.

A variety of hosts are treatable according to the subject methods.Generally such hosts are “mammals” or “mammalian,” where these terms areused broadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), lagomorpha (e.g. rabbits) and primates(e.g., humans, chimpanzees, and monkeys). In many embodiments, the hostswill be humans.

The subject inventions finds use in a number of specific representativeapplications. These applications include: peripheral demineralizingatherectomies; coronary demineralizing atherectomies; and valve/annulardemineralizations. Each of these applications is discussed in greaterdetail separately below.

Peripheral Demineralizing Atherectomy

One type of specific method provided by the subject invention is aperipheral demineralizing atherectomy, in which a calcified targetlesion present in a peripheral vessel, e.g. artery or vein, of thecirculatory system is demineralized. The target lesion may be present inany peripheral vessel, where the subject methods are particularly suitedfor use in the demineralization of lesions that are present in therenal, iliac, femoral, arteries, arteries of the lower extremities, andA-V access sites.

In peripheral demineralizing atherectomy procedures according to thesubject invention, the target calcified lesion is typically flushed witha dissolution solution according to the subject invention for asufficient period of time for the desired demineralization of the targetlesion to occur. The manner in which the target lesion is flushed withthe solution generally depends on the nature of the device that isemployed, as well as the nature of the target lesion. For example, onemay cannulate the vessel on either side of the lesion, with the upstreamcannula being used to introduce the dissolution solution and thedownstream cannula being used to remove solution from the vessel. Inthese embodiments, isolation of the limb comprising the targetperipheral vessel may be indicated, as described above. Alternatively, acatheter device that provides for a substantially sealed localenvironment of the target lesion may be employed to introduce and removethe dissolution solution from the site of the target lesion. Theseprocedures are particularly suited for the treatment of calcified targetlesions that do not substantially occlude the peripheral vessel. Wherethe vessel is substantially, if not completely, occluded by the targetlesion, a device as shown in FIG. 5 may be employed. In FIG. 5, catheter11 has outlet 13 and inlet 15 and is positioned next to the upstreamside of the target lesion 14 that substantially completely occludes thevessel 12. Dissolution fluid is contacted with the target lesion 14 byflowing the dissolution fluid out of the opening 13, preferably underpressure such that the target lesion is contacted with a “jet” ofdissolution fluid. Fluid is also removed via port 15. Importantly, therate of inflow and outflow of fluid from the site of the target lesionis kept substantially constant so that pressure is not substantiallyelevated at the site of the target lesion. This process results in asteady decrease in the size of the target lesion, at least to a pointwhere the lesion does not substantially completely occlude the targetvessel.

The above procedure may be used by itself in a given treatment process,where demineralization of the target lesion is sufficient to achieve thedesired outcome of the particular therapy indicated by the host'scondition. Alternatively, the above procedure may be used in combinationwith additional treatment modalities, including balloon angioplasty;stenting; mechanical atherectomy; bypass and the like, where the subjectmethod of performing a peripheral demineralizing atherectomy serves toprepare the target lesion and vessel for the subsequent treatment. Thus,the subject methods find use in: facilitating the placement of ballooncatheters in narrow, focal, calcified lesions; facilitating theplacement of stents in narrow, focal, calcified lesions; treating totalperipheral vascular occlusions; and facilitating surgical bypass byremoving calcification at proximal and/or distal anastomotic sites orconverting procedures to percutaneous procedures.

Coronary Demineralizing Atherectomy

Another type of specific method provided by the subject invention is acoronary demineralizing atherectomy, in which a calcified target lesionpresent in a vessel associated with the heart, e.g. coronary artery, isdemineralized. The target lesion may be present in any coronary vessel,such as the aorta, coronary arteries, etc.

In coronary demineralizing atherectomy procedures according to thesubject invention, the target calcified lesion is typically flushed witha dissolution solution according to the subject invention for asufficient period of time for the desired demineralization of the targetlesion to occur. The manner in which the target lesion is flushed withthe solution generally depends on the nature of the device that isemployed, as well as the nature of the target lesion. For example, wherethe coronary vessel is not totally occluded by the target lesion, acatheter device that provides for a substantially sealed localenvironment of the target lesion may be employed to introduce and removethe dissolution solution from the site of the target lesion. See e.g.FIG. 2. Where the vessel is substantially, if not completely, occludedby the target lesion, a device as shown in FIG. 5 may be employed. InFIG. 5, catheter 11 has outlet 13 and inlet 15 and is positioned next tothe upstream side of the coronary target lesion 14 that substantiallycompletely occludes the coronary vessel 12. Dissolution fluid iscontacted with the target lesion 14 by flowing the dissolution fluid outof the opening 13, preferably under pressure as described above. Fluidis also removed via port 15. Importantly, the rate of inflow and outflowof fluid from the site of the target lesion is kept substantiallyconstant so that pressure is not substantially elevated at the site ofthe target lesion. This process results in a steady decrease in the sizeof the target lesion, at least to a point where the lesion does notsubstantially completely occlude the target vessel.

The above procedure may be used by itself in a given treatment process,where demineralization of the target lesion is sufficient to achieve thedesired outcome of the particular therapy indicated by the host'scondition. Alternatively, the above procedure may be used in combinationwith additional treatment modalities, including balloon angioplasty;stenting; mechanical atherectomy; coronary artery bypass and the like,where the subject method of performing a coronary demineralizingatherectomy serves to prepare the target lesion and vessel for thesubsequent treatment. Thus, the subject methods find use in:facilitating the placement of balloon catheters in narrow, focal,calcified lesions of coronary vessels; facilitating the placement ofstents in narrow, focal, calcified lesions of coronary vessels; treatingtotal peripheral vascular occlusions in coronary vesels; andfacilitating coronary vessel surgical bypass by removing calcificationin proximal and/or distal anastomotic sites or converting procedures topercutaneous procedures.

Valve/Annulus Demineralization

Yet another application in which the subject methods find use is in thedemineralization of valves and/or annuli, typically those found in theheart or vessels closely associated therewith, e.g. the aortic valve,mitral annuli, etc. In other words, the subject methods are useful indemineralizing valvuloplasties or annuloplasties. The valve/annularstructure that is treatable according to the subject methods may beendogenous to the host or bioprosthetic, i.e. an implant, where theimplant may be a allogenic, xenogeneic, synthetic, etc.

In demineralizing a valve/annular structure according to this particularapplication of the subject invention, the valve or structure having thecalcified lesion present thereon is typically flushed with a dissolutionsolution, as described above. In many embodiments, the local environmentof the valve/annular structure is substantially isolated from theremainder of the host's circulatory system during this flushing step. Avariety of different devices may be employed to flush the structure withthe dissolution solution, including that shown in FIG. 4 describedsupra, that disclosed in U.S. Pat. No. 5,167,628 the disclosure of whichis herein incorporated by reference, and the like.

Demineralizing valvuloplasties and annuloplasties according to thesubject invention can be used to achieve a number of differenttherapeutic goals, including: (a) extension of the useful live ofbioprosthetic implants; (b) enhancing the efficacy of annuloplasty ringplacement; (c) decreasing the calcification of native heart valves,thereby delaying valve replacement; and the like.

Systems

Also provided by the subject invention are systems for use in performingthe subject methods. The systems of the subject invention include atleast a dissolution fluid introductions means, such as the subjectcatheters described above, and a dissolution fluid reservoir capableholding or storing the dissolution fluid just prior to administration tothe local environment of the lesion. In addition, the subject systemswill typically include a means for moving the dissolution fluid throughthe fluid introduction means to the local environment of the lesion,where such means is typically a pump, large syringe, and the like. Thesystem may also conveniently include a means for maintaining thepressure and/or temperature of the dissolution fluid at a desired value.In addition, the subject systems typically include a means for removingfluid from the local environment of the lesion, e.g. a second pumpingmeans or suction means. The above elements of the subject system mayconveniently be present in housing fabricated of a suitable material.

Kits

Also provided are kits for use in performing the subject methods. Thekits typically comprise at least the dissolution fluid to be used in thesubject methods, such as a hydrochloric acid solution or carbonic acidsolution, as described above, where the solution may be present in apressurized and/or climate controlled container so as to preserve thestability of the dissolution solution. For kits that are to be used inmethodologies in which the fluid is flushed through the localenvironment of the lesion, the amount of dissolution fluid present inthe kit ranges from about 1 to 500 liters, usually from about 10 to 200liters and more usually from about 50 to 100 liters. For kits that areto be used in static methodologies, the amount of dissolution fluidpresent in the kit generally ranges from about 100 ml to 1 liter andusually from about 100 ml to 500 ml. Alternatively, the kit may compriseprecursors of the dissolution solution for use in preparing the solutionat the time of use. For example, the precursors may be provided in dryform for mixing with a fluid, e.g. water, at the time of use. Alsopresent in the kit may be a fluid introduction (and even removal) means,as described supra. In addition to the dissolution fluid or precursorsthereof, the kit may further comprise one or more additional fluids (ordry precursors thereof), such as a priming solution, a washing solution,and the like. Finally, the kits will include instructions for practicingthe subject methods, where such instructions may be present on one ormore of the kit components, the kit packaging and/or a kit packageinsert.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental

I. Analysis of Aortic Valve Mineralization

Two human aortic heart valves were removed during routine valvereplacement therapy. These valves were dissected to separate mineralizeddeposits on the valve leaflets. The deposits where strongly adherent tothe valve tissue and were incorporated into the structure of theleaflets as nodules. Both valves had extensive mineralize noduleformation. The nodules were hard and could not be fractured by hand.Contact x-rays were taken to document the extent and distribution of themineralized nodules in the valve tissue. The mineralized areasdemonstrated a radioopacy similar to well mineralized bone.

X-ray diffraction and Fourier Transform Infra Red Spectroscopy (FTIR)were performed using standard procedures (see Constantz, B. R., et al.1995, Science 267: 1796-1799, herein incorporated by reference) on theremoved samples, both directly and following removal of most organicmaterial with sodium hypochlorite (CLOROX bleach). The XRD pattern ofthe mineralized tissue, both with and without the organics removed,showed the characteristic peaks of apatite. The reflections were poorlycrystalline in nature, indicating small crystal size and low levels ofcrystalline order. The FTIR spectrogram of the mineralized tissue, bothwith and without the organics removed, further identify the mineralizeddeposit as apatite that contains substantial carbonate, termed acarbonated apatite (mineral name, dahllite).

Samples were prepared for scanning electron microscopy, using themethods of Constantz, B. R., 1986 (In: Reef Diagenesis, Schroeder, J.,and Puser, B., (eds.), Springer-Verlag). The size of the crystalscomposing the mineralized deposit were less than one micron across. Thesolubility of the crystals in this size range is expected to modify byan order of magnitude due to their increased surface are to volume ratio(see Constantz, B. R., et al., 1986, supra).

The composition of the “calcific deposits” are not calcium orhydroxyapatite as commonly published, rather they are a carbonatedapatite, dahllite, which is expected to be considerably more solublethan hydroxyapatite. Also the size and crystallinity of the crystals ofdahllite comprising these deposits are that of very small, high surfacearea to volume ratio crystallites whose diffraction patterns indicate avery low degree of crystalline order, further increasing theirsolubility.

II. Mineral Dissolution Assays

A. Norian SRS® cement (obtained from Norian Corporation, Cupertino,Calif.) is prepared according to the manufacturer's instructions. Theresultant paste is placed into Teflon mold rings and allowed to set toproduce dahllite disks. The disks are then contacted with the followingsolutions: 0.1 M HCl, 1.0 M HCl, concentrated HCl 0.1 M HCl +0.01 MEDTA, 1.0 M HCl +0.01 M EDTA, concentrated HCl +0.1 M EDTA, 0.1 M H₂SO₄,1.0 M H₂SO₄, 0.1 M H₂SO₄+0.01 M EDTA, 1.0 M H₂SO₄+0.1 M EDTA, 1.0 Mformic acid, concentrated fromic acid, 1.0 M formic acid +0.1 M EDTA,1.0 M acetic acid, concentrated acetic acid, 1.0 M acetic acid and 0.1 MEDTA, 1.0 M succinic acid, 1.0 M succinic acid +0.1 M EDTA; 0.1 Mcarbonic acid; and 1.0 M carbonic acid. A dissolution graph is thenprepared for each solution which plots Ca²⁺ concentration over time. Bycomparing the different dissolution graphs, the solubility of dahllitein different dissolution solutions is compared.

B. Dissolution of Bolus of Dahllite in 0.05N HCl with Various IonicStrengths Using Pump at 69 ml/min.

1. Introduction

Six dissolution experiments were conducted to determine the affect ofionic strength on the dissolution rate of carbonated hydroxyapatite inHCl. According to the Kinetic Salt Effect theory, oppositely chargedions react more slowly as the ionic strength of the solution isincreased because the electrostatic attraction between the reacting ionsis decreased. The object of this experiment was to determine if thetheory holds for the dissolution reaction of carbonated hydroxyapatitewith HCl.

2. Experimental

A Cole-Parmer peristaltic pump (model #7520-35) was used to deliver thedemineralizing 0.05N HCl solution with varying NaCl concentrations tothe sample of carbonated hydroxyapatite (i.e. dahllite),Ca_(8.8)(HPO₄)_(0.7)(PO₄)_(4.5)(CO₃)_(0.7)(OH)_(1.3), in the form of aspherical bolus. In each case, a 100±3 mg bolus (dry weight) ofcarbonated hydroxyapatite was soaked in deionized water until there wasno further weight gain. This weight was taken to be the initial weightof the bolus. The bolus was then transferred to a 12 ml disposableliquid transfer pipette and a peristaltic pump with a rubber stopper onone end of the tubing was attached. Solutions were pumped through thepipette past the bolus at a rate of approximately 69 ml/min in 5 minutetime intervals and the weight of the bolus was measured at the end ofeach interval. The dissolution process was continued until the weight ofthe bolus was less than 5 mg. The NaCl concentrations used were: 0, 5.8(isotonic), 11.6, and 25 g/L.

3. Results

The results of the six dissolution experiments are tabulated below. Atable of the respective half-lives follows. The wet weight of the bolusat t=0 is represented by m(o), and m(t) is the weight at a given timeinterval (m=mass).

TABLE 1 Dissolution of Bolus of 0.05N HCl with Various Ionic Strengthslog[m(t)/m(0)] Time 5.8 g 11.6 g (min) No salt No salt(2) 5.8 g NaClNaCl(2) NaCl 25 g NaCl 0.0 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 5.0−0.0569 −0.0982 −0.0789 −0.0822 −0.1209 −0.1300 10.0 −0.1374 −0.2121−0.1926 −0.1803 −0.2287 −0.3014 15.0 −0.2403 −0.3212 −0.3322 −0.2906−0.3973 −0.5792 20.0 −0.3594 −0.4491 −0.5195 −0.4514 −0.6717 25.0−0.4765 −0.5907 −0.8683 −0.6736 30.0 −0.6273 −0.7788 −1.2653 35.0−0.8154 −1.0740 Half-lives for the Dissolution of Bolus in 0.05N HClHalf- No 5.8 g life Salt NaCl 11.6 g (min) No Salt (2) 5.8 g NaCl (2)NaCl 25 g NaCl 15.8 12.7 11.6 11.8 10.5 8.8

4. Discussion and Conclusion

The half-life data and log[m(t)/m(0)] vs. time show that increasing theionic strength of the solution increases the dissolution rate. Thiscontradicts the Kinetic Salt Effect theory which says that increasingthe ionic strength of a solution decreases the reaction rate betweenoppositely charged ions due to a decrease in electrostatic attractionbetween the ions. In this case, Na⁺ and Cl⁻ ions should theoreticallydecrease the electrostatic attraction between H⁺ and both HPO4²⁻ andPO4³ and slow the rate of dissolution.

C. Dissolution of Bolus in HCl Solutions of Various pH

1. Introduction

Eight sets of dissolution experiments were conducted to determine theaffect of pH on the dissolution rate of carbonated hydroxyapatite inHCl. It was predicted that a decrease in pH (increase in H⁺) shouldincrease the rate of dissolution. In addition, three different methodsof dissolution were used to see how altering the method would affect thedissolution rate.

2. Experimental

For each experiment, a 100+3 mg (dry weight) sample of carbonatedhydroxyapatite, Ca_(8.8)(HPO₄)_(0.7)(PO₄)_(4.5)(CO₃)_(0.7)(OH)_(1.3), inthe form of a spherical bolus was used. The bolus was soaked indeionized water until there was no further weight gain and this weightwas taken to be the initial weight of the bolus. Descriptions of thethree dissolution methods are below. For each set of experiments, ninepH levels were studied.

i. Stirring

For the stirring experiments, the bolus was placed in a beaker with avolume of HCl solution that provided twice the stoichiometric number ofprotons necessary to dissolve the carbonated hydroxyapatite. A stir barof appropriate size was added to the beaker and the solution was stirredon an IKA Labortechnik stir plate on a setting of 6. For eachexperiment, the weight of the bolus was measured at time intervalsappropriate for the pH of the solution used until the weight of thebolus was less than 5 mg. pK was calculated from the slope of the linearregression line for each set of data points using the following formula:

pK=−log[1.533*slope]

ii. Sonication

Sonication experiments employed a Branson Sonifier 450 to deliverultrasound to the HCl solution. Power outputs of 9 Watts, 35 Watts, and53 Watts were used. The solutions were also stirred on an IKA Colorsquidstir plate on a setting of 2 to ensure complete mixing. The bolus wasplaced in a beaker with a volume of HCl solution that provided twice thestoichiometric number of protons necessary to dissolve the it, andweight measurements were made at time intervals appropriate for the pHof the solution until the weight was less than 5 mg. pK was calculatedas it was for the stirring experiments.

iii. Pump

A Cole-Parmer peristaltic pump (model #7520-35) was used to deliver theHCl solution to bolus. The bolus was placed in a 12 ml disposable liquidtransfer pipette and the peristaltic pump with a rubber stopper on oneend of the tubing was attached. Solutions were pumped through thepipette past the bolus at rates of approximately 16 ml/min, 33 ml/min,69 ml/min, and 110 ml/min. Weight measurements were made at appropriatetime intervals until the weight was less than 5 mg, and pK wascalculated as before.

3. Results

The results of the eight sets of dissolution experiments are included inTable 2 below. Graphs were also generated from the observed data. Ratemeasurements for 0.8N, 0.6N and 0.075N were not taken for the sonicationand pump experiments because the slope of the pK vs. pH linearregression line for the stirring experiment was relatively unchanged byincluding these points. Note that a lower pK indicates a fasterdissolution rate.

TABLE 2 pKs Resulting from Dissolution of Bolus with Various HClSolutions and Various Dissolution Methods HCl concen- Soni- Soni- Soni-Pump Pump Pump Pump tration cation cation cation 16 33 69 110 (N) pHStirring 9W 35W 53W ml/min ml/min ml/min ml/min 1.000 0.000 0.71890.1997 0.2634 0.3557 0.4190 0.2710 0.2392 0.2069 0.800 0.097 0.82040.600 0.222 1.1122 0.400 0.398 1.2840 0.7086 0.5689 0.6252 0.8400 0.57170.4875 0.4195 0.200 0.699 1.6950 0.9987 0.8799 0.6922 1.1140 0.98870.7103 0.7015 0.100 1.000 1.8600 1.4970 1.1693 1.1146 1.2390 1.15241.0674 0.8288 0.075 1.125 2.0310 0.050 1.301 1.9440 1.9612 1.4658 1.53641.5020 1.4340 1.2938 0.9704 0.001 2.000 2.2440 2.3607 2.3773 2.06592.3240 1.9982 1.8080 1.6649

4. Discussion and Conclusion

Several conclusions may be drawn from the results of these experiments.First, the positive slopes of the lines on the pK vs. pH graph (notshown) show that a decrease in pH of the solution (increase in H⁺)results in an increase in dissolution rate (decrease in pK) as expected.The dissolution involves H⁺, HPO₄ ²⁻ and PO₄ ²⁻ ions, so it makes sensethat increasing H⁺ should increase the dissolution rate.

Both sonication and the pump gave faster dissolution rates than stirringalone. This is most likely due to the fact that sonication and pumpingprovide better mixing of the solution, effectively removing any layer ofdissolved or repricipitated material from the immediate area surroundingthe bolus.

For the sonication experiments, increasing the ultrasonic powerincreased the rate of dissolution. When ultrasound was used, tinycraters in the surface of the bolus were observed. Increasing theultrasonic power may help dissolution by either increasing the surfacearea due to these craters, increasing the mixing of the solution, orboth. It may also dislodge particles from the surface of the bolus thatare not yet dissolved.

Of the three dissolution methods studied, the pump gave the fastestdissolution rate. The rate consistently increased as the pump flow ratewas increased. The maximum flow rate for the peristaltic pump that wasused was 110 ml/min, but it is anticipated that a faster dissolutionrate may be achieved by using a faster pump. The faster rate may beattributed to the fact that a larger volume of solution (more thandouble the stoichiometric number of protons) must be used with the pump,and that the bolus is always exposed to fresh solution which isequivalent to ultimate mixing. The stream of solution may alsomechanically remove particles from the bolus.

One final observation from the pK vs. pH graph is that differences inrate for the different methods decrease as pH decreases. In other words,rates vary less at pH 0 and vary more at pH 2. Therefore, for solutionsof higher proton concentration, the rate of dissolution is lessdependent on the method employed.

D. Dissolution of Bolus in HCl Solutions using Ultrasound

1. Introduction.

Six sets of dissolution experiments were conducted to determine theeffect of Ultrasound on the dissolution rate of carbonatedhydroxyapatite in HCl. It was predicted that an increase in ultrasonicpower should increase the rate of dissolution due to an increase inmixing of the solution.

2. Experimental

For each experiment, a 100±3 mg (dry weight) sample of carbonatedhydroxyapatite, Ca_(8.8)(HPO₄)_(0.7)(PO₄)_(4.5)(CO₃)_(0.7)(OH)_(1.3), inthe form of a spherical bolus was used. The bolus was soaked indeionized water until there was no further weight gain and this weightwas taken to be the initial weight of the bolus. The bolus was placed ina beaker with a volume of HCl solution that provided twice thestoichiometric number of protons necessary to dissolve it, and a BransonSonifier 450 was employed to deliver ultrasound to the solution. VariousHCl solutions were employed. The 0.1N, 0.05N, and 0.01N HCl solutionswere made isotonic (300 mOsmol) with NaCl. Power outputs of 9 Watts, 35Watts, and 53 Watts were used. The solutions were also stirred on an IKAColorsquid stir plate on a setting of 2 to ensure complete mixing.Weight measurements were made at time intervals appropriate for the pHof the solution until the weight was less than 5 mg. pK was calculatedfrom the slope of the linear regression line for each set of data pointsusing the following formula:

pK=−log[1.533*slope]

3. Results

The results of the six sets of dissolution experiments are tabulatedbelow in Table 3. Note that a lower pK indicates a faster dissolutionrate.

TABLE 3 Ultrasonic Power 1N 0.4N 0.2N 0.1N 0.05N 0.01N (Watts) HCl HClHCl HCl HCl HCl pKs Resulting from Dissolution of Bolus Using Ultrasound9 0.1997 0.7086 0.9987 1.497 1.9612 2.3607 35 0.2634 0.5689 0.87991.1693 1.4658 2.3773 53 0.3557 0.6252 0.6922 1.1146 1.5364 2.0659Half-lives (in min) Resulting from Dissolution of Bolus Using Ultrasound9 1.1 3.6 6.8 18.5 48.3 130.5 35 1.2 2.2 4.3 8.9 15.6 130.7 53 1.2 2.6 37.9 19.3 64.2

4. Discussion and Conclusion

The half-life data table shows that when the ultrasonic power wasincreased from 9 Watts to 35 Watts, the rate of dissolution increasedfor all solutions except 1N HCl and 0.01N HCl for which the ratesremained relatively unchanged. The 1N HCl solution dissolves the bolusso quickly that any minor rate changes are difficult to observe. It isunclear why there was no observable increase in dissolution rate for the0.01N solution. When the ultrasonic power was increased to 53 Watts,dissolution rates increased for all solutions except 1N and 0.4N, forwhich rates remained relatively unchanged, and 0.05N for which the ratedecreased slightly. The results indicate that increasing the ultrasonicpower increased the dissolution rate except when the rate is already sofast that minor changes are difficult to observe.

E. Dissolution of Bolus in HCl Solutions Using Pump

1. Introduction

Six sets of dissolution experiments were conducted to determine theeffect of pump flow rate on the dissolution rate of carbonatedhydroxyapatite in HCl. It was predicted that an increase in flow rateshould increase the rate of dissolution due to an increase in exposureto protons.

2. Experimental

For each experiment, a 100±3 mg (dry weight) sample of carbonatedhydroxyapatite, Ca_(8.8)(HPO₄)_(0.7)(PO₄)_(4.5)(CO₃)_(0.7)(OH)_(1.3), inthe form of a spherical bolus was used. The bolus was soaked indeionized water until there was no further weight gain and this weightwas taken to be the initial weight of the bolus. The bolus was placed ina 12 ml disposable liquid transfer pipette and a Cole-Parmer peristalticpump (model #7520-35) with a rubber stopper on one end of the tubing wasattached. Various HCl solutions were pumped through the pipette past thebolus at rates of approximately 16 ml/min, 33 ml/min, 69 ml/min, and 110ml/min. The 0.1N, 0.05N, and 0.01N HCl solutions were made isotonic (300mOsmol) with NaCl. Weight measurements were made at time intervalsappropriate for the pH of the solution until the weight was less than 5mg. pK was calculated from the slope of the linear regression line foreach set of data points using the following formula:

pK=−log[1.533*slope]

3. Results

The results of the six sets of dissolution experiments are tabulatedbelow. Note that a lower pK indicates a faster dissolution rate.

TABLE 4 Pump Flow Rate 1N 0.4N 0.2N 0.1N 0.05N 0.01N (ml/min) HCl HClHCl HCl HCl HCl pKs Resulting from Dissolution of Bolus Using Pump 160.4190 0.8400 1.1140 1.2390 1.5020 2.3240 33 0.2710 0.5717 0.9887 1.15241.4340 1.9982 69 0.2392 0.4850 0.7103 1.0674 1.2938 1.8084 110 0.20690.4195 0.7015 0.8288 0.9704 1.6649 Half-lives (in min) Resulting fromDissolution of Bolus Using Pump 16 1.5 3.9 7.6 10.0 21.1 110.5 33 1.12.2 5.5 9.2 15.3 61.5 69 1.0 1.5 3.0 6.7 11.6 38.8 110 0.8 1.6 2.8 4.86.4 29.2

4. Discussion and Conclusion

The data show an obvious increase in dissolution rate as the pump speedis increased. 110 ml/min was the fastest flow rate that could beattained with this pump, however it is likely that the dissolution ratewould continue to increase with a faster pump. The increase in rate maybe attributed to the increase in exposure of the bolus to protons.Mechanical removal of surface particles may also play a role.

III. Demineralizing a Calcified Aorta

A. Materials

A human heart with an attached aorta and corotid artery branches wasobtained and characterized flouroscopically for the presence ofmineralization. The mineralized deposits are radio-opaque and arewell-established to be the calcium phosphate mineral carbonated apatite(dahllite) [see Tomasic 1994 In: Brown and Constantz, Hydroxyapatite andRelated Materials CRC Press]. Physical manipulation of the tissueindicated that the mineral makes the vessel rigid and the walls of thevessel are hard. Extensive mineralization was seen in the aorta and thethree corotid artery branches. Two of the three side branches of thebrachial-cephalic corotid artery were completely occluded withmineralization. The other two corotid artery branches were partiallyoccluded with mineralization.

B. Experimental Set-Up

The distal and proximal ends of the aorta were cannulated and tubing wasattatched. The distal outflowing tube has a “Y” to allow the exfluentsolution to flow into two different collection traps: one fordemineralizing solution, the other for saline wash. The reason for thisdesign is that the calcium concentration is measured in the exfluentdemineralizing solution so it needs to be isolated from the occasionalsaline wash to remove contrast media. An infusion catheter was placedthrough the wall of the proximal tubing and advanced into the aorta tojust proximal of the brachio-cephalic corotid branch point. The exfluentports of the unoccluded corotid arteries and the distal aotric tube wereclipped off with hemostats and contrast media was infused into theinfusion catheter under fluoroscopy, filling the aorta with radio-opaquecontrast media. The extent of occlusion was quantified fluoroscopically.The hemeostats were then unclipped and the system was flushed withsaline.

C. Demineralization

4 liters of 1N hydrochloric acid with 0.25 mole/liter sodim chlorideconcentration were infused through the infusion catheter by drawing thedemineralizing solution into 60 ml syringes with lure-lock cannulae,attaching them to the infusion catheter and injecting at a rate rangingbetween 125 and 250 ml/minute. Four successive infusion segments wereperformed:

0-5 minutes

5-10 minutes

10-15 minutes

15-20 minutes

Between each five minute infusion the system was flushed with saline,the open exfluent ports were clipped with hemostats, radio-opaquecontrast media was infused and the extent of mineralization quantifiedfluoroscopically. Following this evalution the hemostats were unclippedand the system flushed with saline and the next infusion begun.

D. Results

By the end of the experiment when all four liters of demineralizingsolution had been infused, all three totally occluded sub-branches ofthe brachiocephalic corotids artery had been opened and solution flowedfrom their distal ports.

1. 0-5 Minutes (Approximately 550 ml)

The solution flowed out of the two partially occluded corotid arteriesand the distal aortic tube was clipped off. About 2 minutes into theinfusion, solution began dripping from the totally occludedbrachio-cephalic segments. When the collected exfluent demineralizingsolution was observed, removed solids were collected in a 50 mlcentrifuge vial; approximately 20 ccs of solid white material waspresent. The radio-contrast at 5 minutes showed the occluded arteriesopening up and the lumen of all the arteries opening. The general extentof mineralization was also noticeably diminished.

2. 5-10 Minutes (Approximately 1 Liter)

Now the most open corotid artery was clipped off, the partially occludedcorotid artery was half clipped off, allowing limited out flow and thedistal aortic tube was totally clipped-off. Flow progressively increasedfrom the brachio-corotid arteries and two of the three sub-segmentsbegan flowing substantially, as did the partially occluded third corotidartery. Radio-contrast imaging at 10 minutes corroborated the flowobservations, showing the arterial lumen had considerably opened toallow flow.

3. 10-15 Minutes (Approximately 1 Liter)

Now both open corotid arteries were clipped off, allowing limited outflow through the brachial-cephalic corotid artery and the distal aortictube was totally clipped-off. Flow progressively increased from thebrachio-corotid artery and two of the three sub-segments began flowingsubstantially and third sub-segment began flowing somewhat.Radio-contrast imaging at 15 minutes corroborated the flow observations,snowing the arterial lumen had considerably opened to allow flow.

4. 15-20 Minutes (Approximately 1.5 Liters)

Now both open corotid arteries were clipped off as well as the twoflowing brachio-cephalic sub-segments, allowing limited out flow throughone the brachial-cephalic sub-segment that was most occluded at thebeginning and was still only flowing in a restricted fashion. The distalaortic tube was totally clipped-off. Flow progressively increased fromthe brachio-corotid sub-segment and began flowing to the extent that theflow squirted off the table onto the floor. Radio-contrast imaging at 20minutes treatment corroborated the flow observations, showing thearterial lumen had considerably opened to allow flow.

E. Conclusion

By the end of the experiment, a heavily calcified aorta and corotid treewas substantially demineralized and flow re-established. The arotachanged from being hard to soft and resiltiant to the touch. Thevascular tissue showed no mechanical loss of strength of flexiblebehavior.

IV. Demineralizing Human Cadaveric Leg Arteries—Evaluation ofDemineralizing Capabilities

The decalcification properties of the demineralizing solution areexamined by applying it to in vitro human cadaver vessels. The resultsare qualitatively analyzed by intravascular ultrasound and contact x-rayimaging while quantitative analysis is performed by atomic absorptionspectroscopy.

A. Introduction

In order to evaluate the capabilities of the demineralizing solution,cadaver arteries are perfused with demineralizing solution. Sections ofleg vessels from 12 cadavers (fermoral to tibial artery) are obtainedwith a wide range of calcifications. Intravascular ultrasound andcontact x-rays are utilized to qualitatively assess the location andamount of calcification in each vessel prior to perfusion.

B. Demineralizing Procedure

The method for demineralizing the cadaver vessels is designed torealistically simulate the envisioned clinical use of the demineralizingsolution. In order to model this, segments of the vessels are sutured totubing in a water bath and perfused with the one of three differentdemineralizing solutions for 2 minutes using a peristaltic pump at aflow rate of 50 ml/minute. The demineralizing solutions are: (1)Solution A=1.0 N HCl+0.25 M NaCl; (2) Solution B=.0.5 N HCl+0.25M NaCl;and (3) Solution C=0.1 N HCl+0.05 M NaCl. The demineralizing solutionpumped through the vessel is captured in order to quantitate the amountof decalcification.

C. Calcification Analysis

Before and after the demineralizing procedure, intravascular ultrasoundis performed with automatic pullback through the vessel and imagingsequences stored to videotape. This ultrasound analysis preserves avideo record of the entire length of the vessel which may bequalitatively analyzed for calcium content before and after theprocedure. Contact x-rays are also performed for the same purpose andprovides a silhouette of the vessel segment. Quantitativedecalcification data is obtained by utilizing an atomic absorptionspectrometer to measure the calcium concentration in the demineralizingsolution captured. By using the stoichiometric formula for theatherosclerotic calcifications, the mass of the material actuallyremoved is calculated.

EXAMPLE V Infusing Demineralizng Solution Through the Murine AbdominalAorta—Vascular Response to Demineralizing Solution

The following protocol is designed to determine the in vivo cellularresponse of a vessel to a demineralizing solution developed to treatcalcified lesions of the cardiovascular system. This determination ofresponse is accomplished by applying the solution utilized for thedecalcification procedure to non-calcified, in vivo rabbit aorta inorder to examine the histological response. The demineralizing solutionis designed to minimize the cellular damage while still demineralizingvessels effectively; thus, it is believed with a reasonable expectationof success that there will be little or no damage to the vessel tissue.

A. Introduction

In order to evaluate vascular response to the demineralizing solution,25˜male, New Zealand White rabbits (>4 kg) have a segment of their aortaexposed to either a demineralizing solution or a saline control. Thesegment is isolated from blood by using two standard catheter balloonswith a lumen through which the solution may be perfused. The rabbits arethen sacrificed after waiting for an established period of time andhistology is performed on the aortic segments.

For the initial phase, 27 rabbits are treated, and 3 differentconcentrations of demineralizing solution are tested along with a salinecontrol. Solutions A, B and C are the same as those employed in ExampleIV above. The concentration range is chosen with significant chemicaldifferences so that a concentration dependent response is expected.Rabbits from each group are sacrificed at 1, 7, 30 and 90 days.Additionally, 3 rabbits are perfused with saline as controls andsacrificed at 1 (1 rabbit) and 7 days (2 rabbits).

B. Surgical Procedure

On the appropriate day, the selected rabbits are prepared for surgery.The surgical procedure includes the standard anesthesia and physiologicmeasurement techniques utilized in rabbit studies.

Following preparation, the right or left carotid artery is exposed andcarefully incised, and a 5 Fr sheath is inserted. The right or leftfemoral artery is then exposed and carefully incised, and a 4 or 5 Frsheath inserted. After 1000U heparin injection (IV), a Schwan-Ganzcatheter isnserted via the right or left carotid artery to the abdominalaorta just distal to the renal artery branch under fluoroscopicguidance. A second Schwan-Ganz catheter is inserted via the right orleft femoral artery into the abdominal aorta just proximal of thebifurcation of the iliac arteries under fluoroscopic guidance.

Balloon position is monitored angiographically, providing approximately2 cm section of abdominal aorta for exposure to the demineralizingsolution. Following balloon inflation, the isolated environment isrendered substantially bloods by flushing with heparanized saline. Next,sterile demineralizing solution at the scheduled concentration (or thesaline control solution) is pumped into the carotid catheter and removedthrough the femoral catheter by means of a peristaltic pump at a flowrate of 50 ml/minute for 2 minutes. Following this, the region isflushed by pumping 0.9% NaCl through the system for 20 seconds.

C. Euthanasia and Histological Analysis

Rabbits are euthanized at the scheduled postoperative time by a standardintravenous injection. Immediately following euthanasia, the abdominalaorta is harvested and pressure fixed with 10% formalin. Subsequent tofixation, vessels are embedded, sectioned, and stained by a qualifiedpathologist. Hemotoxylin and eosin (H&E) are used to examine theendothelial and vessel cells, and a trichrome stain is used to examinethe connective and collagenous tissue elements. Sections of vesselbetween catheters (experimental condition) are compared with sectionsadjacent to the angioplasty balloons (control condition) for thedifferent concentrations of demineralizing solution and controlsolution. Also, the kidneys, lungs, and liver are frozen for toxicitytesting at a later time.

VI. Human Aortic Valve Tests in Vitro

Calcified aortic heart valves are removed operatively during valvereplacement surgery and used as an in vitro test system to optimizemethods of demineralization. For these studies, a comparison ofdifferent acidic treatment solutions is performed. In addition, theacidic treatment solutions are contacted with the calcified aorticvalves for varying periods of time. The following solutions andconditions are examined:

0.1 M HCl, 1.0 M H₂SO₄ + 0.1 M EDTA, 1.0 M HCl, 1.0 M formic acid,concentrated HCl, concentrated formic acid, 0.1 M HCl + 0.01 M EDTA, 1.0M formic acid + 0.1 M EDTA, 1.0 M HCl + 0.01 M EDTA, 1.0 M acetic acid,concentrated concentrated HCI + 0.1 M EDTA, acetic acid, 0.1 M H₂SO₄,1.0 M acetic acid and 0.1 M EDTA, 1.0 M H₂SO₄, 1.0 M succinic acid, 0.1M H₂SO₄ + 0.01 M EDTA, 1.0 M succinic acid + 0.1 M EDTA.

Explanted valves are weighed after thorough washing. A flow throughsystem comprising the valves are then prepared and each solution isflowed through the explanted valve. Weight loss is measured at 5, 10,30, 60, 90 and 120 minutes of incubation in the acidic treatmentsolution. This experiment is repeated, applying ultrasonic energy at 25MHz during the flow through period.

During the course of treatment, calcium and phosphate concentrationreleased from the test valves are measured, and rates ofdemineralization are determined. The valves are evaluated for the extentof remaining biomineralization and the extent of tissue damage (if any)after each experimental protocol. Physiological function tests are alsoperformed following the contact with the acidic treatment solution.

The following parameters are evaluated and optimized alone or incombination: pH, and sonic power. The carbonated apatite ofbiomineralized heart valve tissue is more soluble under acidicconditions. The pH ranges to be evaluated are below about 7.0, typicallybelow about 4.0, and optimally about 1.0. Sonic power acceleratesreactions in solution. Sound frequencies in the range of 20 kHz to 100kHz are evaluated, typically about 25 kHz.

Prior to and after decalcification, valve insuffinciency is evaluatedusing a pulse duplicator that measures pressure drop across the valve.In addition, flow rate through the valve is measured to obtaininformation regarding the cross-sectional area of the valve.

VII. Human Aortic Valve Tests in a Porcine Model

The most common causes of pure aortic stenosis are calcification ofbicuspid valves, commissural fusion, degenerative calcification oftricuspid valves, cuspid fibrosis, and postinflammatory calcification ofrheumatic origin. Calcified valves removed from human patients aretransplanted into pigs. The animals are allowed to recover, and are thensubjected to demineralization therapy with the acidic treatmentsolution. Devices for applying the acidic treatment solution to thestenotic aortic valves are made to either apply to a beating heart or astopped heart. Devices applied to the beating heart will introduce theacidic treatment solution at a specific temperature and pH inconjunction with sonic power. Devices applied to the stopped heart, orto a bypassed heart which is still beating, will isolate the aorticvalve region form the blood stream and circulate and cycledemineralizing solution through the aortic valve region.

The surgeon attempts to create grain boundary separations betweenindividual grains of the carbonated apatite (dahllite), which composesthe calcified tissue. Acidic solutions preferentially dissolve thecalcium phosphate mineral at grain boundaries. Combined with ultrasonicpower, this serves to loosen individual grains without having todissolve the entire grain. Loose grains are removed with the circulatingsolution. Organic matrices entombed within the mineralized deposit mayshield the mineral phase from the acidic treatment solution,necessitating solutions that are efficient in removing elements of anorganic matrix from the grain boundary regions. Thus, one can supplementthe acidic treatment solution with proteases, surfactants, detergents,oxidants and the like, at concentration sufficient to remove organicmatrix without undue damage to the tissue under treatment.Alternatively, the supplements can be provided in one or more individualsolutions and alternated with the acidic treatment solution. The removalof the organic matrix exposes the mineral to subsequent treatment withacidic treatment solution. Various solutions can be suction-pumpedthrough the treated region through tubing. In this embodiment, bothin-current and out-current flows are present. The out-current flowcarries the cycled solutions, the dissolved ions from the mineral withorganic debris, and loose pieces of mineralized deposits which becomedislodged from the attached mineralized mass before dissolution of themineral deposit is complete. Different solutions are cycled through thetest region from a single site outside the body. Progression ofdemineralization is monitored using standard echocardiographic methods.

Following the above protocol, the animals are sacrificed and the heartvalves are subjected to histological examination and further assays formineral content.

VIII. Formulations

(A) Solution A=1.0 N HCl+0.25 M NaCl.

(B) Solution B=0.5 N HCl+0.25M NaCl.

(C) Solution C=0.1 N HCl+0.05 M NaCl.

(D) A suitable formulation for acidic treatment under a constant flowrate comprises:

Formic acid (concentrated) . . . 0.10%

Sodium dodecyl sulfate (SDS) . . . 0.1%

H₂O . . . qs . . . 100%

(E) An alternative formulation for acidic treatment under a constantflow rate comprises:

HCl (concentrated) . . . 0.10%

EDTA . . . 0.1%

H₂O . . . qs . . . 100%

(F) An alternative formulation for acidic treatment under a constantflow rate comprises:

Phosphoric acid (concentrated) . . . 10%

H₂O . . . qs . . . 100%

(G) An alternative formulation for acidic treatment under a constantflow rate comprises:

Sulfuric acid (concentrated) . . . 10%

H₂O . . . qs . . . 100%

(H) An alternative formulation for acidic treatment under a slower rateor under static conditions comprises:

Tris HCl . . . 0.1 M

pH adjusted to 4.2 with concentrated HCl.

IX. The Sheep Model

To evaluate the efficacy of an acidic treatment solution in vivo, asheep model is utilized. In this model, porcine aortic valved conduitsare treated with 0.625% glutaraldehyde in vitro, and transplanted intothe descending thoracic aorta in juvenile sheep (see Chanda, J., et al.,1997, Biomaterials 18:1317-1321, herein incorporated by reference). Thecalcification of the transplanted porcine valves are then analyzed bygross inspection, radiography, light, transmission, and surface scanningelectron microscopy, or calcium analysis by absorption spectroscopy canbe performed (see Schoen, F. J., et al., 1994, J. Thorac. Cardiovasc.Surg. 108:880-887). Any tissue damaged is also assessed by lightmicroscopy.

A standard open chest surgical procedure is employed where the animal isput on cardiopulmonary bypass and the device shown in FIG. 4 is insertedinto the aorta and into the valve. The valve is then contacted withSolution A describe supra, and the demineralization of the calcifiedvalve is determined.

X. Cadaver Study

Human coronary arteries are harvested from heart transplants. Contactx-rays are taken to document the extent and distribution of themineralized lesions in the harvested arteries. The arteries are ligatedat either end to 5 mm polypropylene tubing and a pumping means isemployed to produce a flow through device. A carbonic acid solutionhaving a pH of 2.5 to 3.0 is flowed through the artery for a period of20 minutes. During this period, intravascular ultrasound (IVUS) isemployed to monitor the lesion in real time. The carbonic acid solutionthat has passed through the artery is collected and analyzed fordissolved calcium content using standard analytical methods. A graph isplotted of the mineral content of the collected solution vs. time.Following treatment, a second contact X-ray is taken to assess theextent of the mineralized lesion remaining in the artery. Finally, anash weight test is performed of the remaining mineral. It is observed inthe above assays that flowing a carbonic acid solution through thecoronary artery reduces the mineral content of the lesion in the artery.Additional arteries are assayed according to the above methods, with thedifference being that flow through times of 30, 60, 90 and 120 minutesare employed.

XI. Rabbit Study

To compare the effect of using a carbonic acid dissolution solutionaccording to the subject invention with the effect of mechanicaldebridement as produced by a balloon catheter on arterial tissue, thefollowing experiments are performed. Three groups of New Zealand whiterabbits are formed, 5 rabbits per group. In each group, the rightfemoral and iliac arteries are treated with a first treatment method(either dissolution fluid or mechanic debridement) and the left iliac orfemoral arteries are treated with another method or left untreated toserve as a control. The following table summarizes the treatmentprotocols.

Group Right Arteries Left Arteries 1 solution (−) control 2 solution (+)mechanical debridement 3 (+) mechanical (−) control debridement

Following 1, 7, 30 & 90 days, the animals are sacrificed and the illiacand femoral arteries harvested for analysis. The arteries arehistographically analyzed for evidence of mechanical insult response andthe presence of smooth epithelial cells. It is expected that thesolution treated arteries appear similar to the control arteries, whilethe arteries subject to mechanical debridement show a mechanical insultresponse.

It is evident from the above results and discussion that improvedmethods of treating a host suffering from a vascular diseasecharacterized by the presence of a calcified lesion are provided. Thesubject methods can be performed in a minimally invasive manner, therebyproviding all of the advantages attendant therewith, such as reducedtrauma to the patient and faster healing times. As the subject methodsdo not mechanically damage the vascular tissue of the patient,complications such a restinosis are avoided. Furthermore, the subjectmethods provide an effective and efficient means for removing calcifiedlesions from vascular tissue. In addition, the subject methods can beused in conjunction with additional treatment modalities, therebyimproving the outcome realized with such modalities. As such, thesubject invention represents a significant contribution to the art.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A method for treating a host suffering from avascular disease, said method comprising: flushing a vascular lesionwith an acidic dissolution fluid having a pH that does not exceed about3 to treat said host.
 2. The method according to claim 1, wherein saidmethod further comprises applying energy to said calcified lesion in amanner sufficient to breakup said lesion into particles.
 3. The methodaccording to claim 1, wherein said method further comprises rendering alocal environment of said vascular lesion substantially bloodless. 4.The method according to claim 1, wherein said acidic dissolution fluidcomprises an organic or inorganic acid.
 5. The method according to claim4, wherein said acidic dissolution fluid is a hydrochloric acidsolution.
 6. The method according to claim 4, wherein said acidicdissolution fluid is hypertonic.
 7. A system for flushing a vasculartissue site with a dissolution fluid, said system comprising: (a) acatheter comprising an acidic dissolution fluid introduction lumencapable of delivering fluid to said vascular tissue site and a fluidremoval lumen capable of removing fluid and lesion debris from saidvascular tissue site, wherein said catheter is in fluid communicationwith an acidic dissolution fluid source comprising an acidic dissolutionfluid having a pH that does not exceed about 3; (b) a first pumpingmeans operatively linked to said fluid introduction lumen in a mannersufficient such that said first pumping means forces fluid out of thedistal end of said fluid introduction lumen; and (c) a second pumpingmeans operatively linked to said fluid removal lumen in a mannersufficient such that said second pumping means sucks fluid into thedistal end of said fluid removal lumen.